Qihong polysaccharide KBPW1 with antitumor activity and its application in preparing antitumor drugs

Abstract

The present invention discloses a Qihong polysaccharide KBPW1 with anti-tumor activity and its application in the preparation of anti-tumor drugs, relating to the technical field of plant polysaccharide active substances and their applications. The Qihong polysaccharide KBPW1 has a repeatable primary structural unit containing eight glycosidic bonds: →4)-β-D-Glcp-(1→, α-L-Araf-(1→, →3)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,5)-β-L-Araf-(1→, β-D-Glcp-(1→, →4,6)-β-D-Glcp-(1→, →6)-β-D-Glcp-(1→), and is composed of arabinose, galactose, and glucose. The molar ratio of arabinose, galactose, and glucose is 2.62:1.02:6.36; the relative molecular mass is 3.8×10 3 Da. Beneficial Effects: As an active anticancer ingredient, it can significantly inhibit the growth, proliferation, migration and invasion of colorectal cancer CT26WT cells, reduce the tumor volume and weight of CT26WT tumor-bearing mice, and has minimal toxicity and side effects on the body. It has good biocompatibility, strong specificity, and low toxicity and side effects. It can make up for the shortcomings of traditional cancer treatments and can be used in the development of new anticancer drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of plant polysaccharide active substances and their applications, and in particular to Qihong polysaccharide KBPW1 with anti-tumor activity and its application in the preparation of anti-tumor drugs. Background Art

[0002] Cancer, a major global public health issue, remains one of the most serious diseases posing a serious threat to human health. In recent years, the incidence and mortality rates of cancer have steadily increased. According to the 2020 Global Cancer Statistics Report, my country experiences 4.57 million new cancer cases and 3 million deaths annually, accounting for 23.7% of the global total. Both the number of new cases and deaths each year far exceeds that of other countries. Cancer treatment has long been a thorny issue in the medical field. Conventional treatments often combine surgery with chemotherapy and radiotherapy, but these approaches are ineffective. Traditional anti-cancer drugs have significant side effects. While they kill tumor cells, they also damage normal cells and immune cells, leading to damage to multiple tissues and organs, a poor prognosis, and rapid malignant progression. Therefore, the development of new, highly targeted cancer treatments and drugs is urgently needed to improve the quality and survival of cancer patients. The search for safe, effective, and specific natural anti-cancer drugs has become a growing goal for researchers in China and abroad.

[0003] Keemun black tea (hereinafter referred to as Keemun) is one of the world's three most fragrant black teas and one of my country's ten famous teas. Primarily produced in Qimen County, Huangshan City, Anhui Province, my country, Keemun is known worldwide for its unique aroma, which resembles flowers, fruits, and honey—the "Qimen fragrance"—and is widely praised and favored by consumers. Documented medicinal uses of Keemun tea date back over a century, and long-term consumption has been shown to have numerous health benefits, including stomach nourishment, heat-clearing, diuretic, anti-inflammatory, antibacterial, refreshing, anti-aging, and cardiovascular disease prevention. Modern pharmacological research indicates that tea polysaccharides are key components in tea's diverse biological activities, exhibiting significant therapeutic effects in anti-tumor, immune-enhancing, and antioxidant activities. The chemical structure of tea polysaccharides is the material basis for their biological activity. However, due to the unique fermentation process and production location, the structure and physiological activities of Keemun's polysaccharides differ from those of other teas, worthy of further investigation.

[0004] At present, the main active components of Qihong polysaccharide are still unclear, and its active substance basis and efficacy are yet to be clarified. At the same time, the fine chemical structure of Qihong anti-cancer active polysaccharide has not been reported, and there is a lack of suitable extraction and purification processes to prepare Qihong polysaccharide with clear chemical structure and uniform composition for anti-cancer activity. These have restricted the research and development of new anti-cancer drugs of Qihong polysaccharide.

[0005] Chinese patent application publication number CN105859903A discloses a Glehnia littoralis polysaccharide, its preparation method, and applications, relating to the field of polysaccharide technology. The Glehnia littoralis polysaccharide has a molecular weight of 26.3 kDa; its monosaccharide composition and molar ratio are: rhamnose: galactose: glucose = 2.05:1.00:7.06; and provides the primary structural repeating unit of the Glehnia littoralis polysaccharide. The Glehnia littoralis polysaccharide has the advantages of a simple extraction process, high component purity, excellent anti-tumor and antioxidant activities, and minimal toxic side effects. However, the patent does not disclose the Qihong polysaccharide KBPW1 of the present invention and its applications. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to provide a Qihong polysaccharide with anti-tumor activity.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention provides a Qihong polysaccharide KBPW1 with anti-tumor activity, wherein the repeatable primary structural unit of the Qihong polysaccharide KBPW1 contains eight glycosidic bonds: (1→4)-β-D-Glcp-(1→, α-L-Araf-(1→, →3)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,5)-β-L-Araf-(1→, β-D-Glcp-(1→, →4,6)-β-D-Glcp-(1→, →6)-β-D-Glcp-(1→); the connection mode of the glycosidic bonds is shown in Formula 1:

[0009]

[0010] Beneficial effects: The Qihong polysaccharide KBPW1 provided by the present invention has a novel structure, a clear chemical structure, and a uniform composition. In vitro cell and in vivo animal experiments have shown that the Qihong polysaccharide KBTP-1 has a good anti-tumor effect.

[0011] Preferably, the relative molecular mass of the Qihong polysaccharide KBPW1 is 3.8×10 3 Da.

[0012] Preferably, the Qihong polysaccharide KBPW1 consists of arabinose, galactose and glucose, and the molar ratio of arabinose, galactose and glucose is 2.62:1.02:6.36.

[0013] The second aspect of the present invention provides the use of the Qihong polysaccharide KBPW1 in the preparation of anti-tumor drugs.

[0014] Preferably, the tumor comprises colorectal cancer (CRC), bile duct cancer, gastric cancer, glioblastoma (GBM), leiomyosarcoma, melanoma, lung cancer, neuroblastoma (NB), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, breast cancer or any combination thereof.

[0015] The third aspect of the present invention provides an anti-tumor pharmaceutical composition, the ingredients of which include the above-mentioned Qihong polysaccharide KBPW1.

[0016] Preferably, the composition further includes one or more pharmaceutically acceptable carriers.

[0017] Preferably, the pharmaceutically acceptable carrier is a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.

[0018] Preferably, the pharmaceutically acceptable carrier is a diluent.

[0019] Preferably, the oral dosage form of the pharmaceutical composition includes tablets, capsules, lozenges, oral liquids, granules, pills or powders.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are embodied in the following aspects:

[0021] 1. The Qihong polysaccharide KBPW1 provided by the present invention has a novel structure, a clear chemical structure, and a uniform composition. Through in vitro cell and in vivo animal experiments, it is proved that Qihong polysaccharide KBPW1 has a good anti-tumor effect, and can also alleviate the weight loss caused by 5-Fu, improve the immune organ index and liver function of mice, and make up for the shortcomings of existing chemical anticancer drugs in terms of large side effects on the body.

[0022] 2. The Qihong polysaccharide KBPW1 provided by the present invention can significantly inhibit the growth, proliferation, migration and invasion of colorectal cancer CT26 WT cells. In animal experiments, the tumor growth of tumor-bearing mice given the polysaccharide was effectively inhibited, with a tumor inhibition rate of up to 71.26%. Even when the administration concentration reached a high concentration of 200 mg / kg, the life characteristics of the tumor-bearing mice were still significantly better than those of the control group that was not given the polysaccharide and was given the positive drug 5-FU.

[0023] 3. The Qihong polysaccharide of the present invention has good anti-tumor activity both in vitro and in vivo, and can significantly inhibit the growth and proliferation of colorectal cancer without any toxic side effects. Currently, no reports on the anti-tumor activity of this Qihong polysaccharide have been found.

[0024] 4. The present invention provides a new Qihong polysaccharide compound with a clear chemical structure. According to high performance liquid chromatography (HPGPC) analysis, the Qihong polysaccharide prepared by the method of the present invention has only one absorption peak and a molecular weight of 3.8×103 Da has strong anti-tumor activity and can be used as a new active ingredient in the preparation of anti-colorectal cancer drugs.

[0025] 5. The method of the present invention has high efficiency in preparing active polysaccharides and stable activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Graph showing the effects of material-liquid ratio (A), temperature (B) and time (C) on the yield of Qihong polysaccharide in Example 1 of the present invention;

[0027] Figure 2 This is a response surface analysis diagram of the factors designed for the preparation process of Qihong polysaccharide in Example 1 of the present invention; AB: material-liquid ratio and temperature; CD: material-liquid ratio and time; EF: time and temperature;

[0028] Figure 3 The elution curves of Qihong polysaccharide KBPW1 in Example 2 of the present invention are shown in FIG. A. The elution curve of Qihong polysaccharide KBPW1 on a DEAE-Sepharose Fast Flow ion exchange chromatography column; and B. The elution curve of Qihong polysaccharide KBPW1 on a Sephadex G-100 gel column.

[0029] Figure 4 The ultraviolet (UV) full wavelength scanning diagram (A) and HPGPC molecular weight chromatogram (B) of Qihong polysaccharide KBPW1 of Example 3 of the present invention;

[0030] Figure 5 This is a Fourier transform infrared spectrum (FT-IR) of the Qihong polysaccharide KBPW1 of Example 3 of the present invention;

[0031] Figure 6 This is a scanning electron microscope (SEM) image of the Qihong polysaccharide KBPW1 of Example 3 of the present invention;

[0032] Figure 7 This is an ion chromatogram (IC) of the monosaccharide composition of the Qihong polysaccharide KBPW1 of Example 3 of the present invention;

[0033] Figure 8 This is a GC-MS graph showing the glycosidic bond type of the Qihong polysaccharide KBPW1 of Example 4 of the present invention;

[0034] Figure 9 is the NMR spectrum of Qihong polysaccharide KBPW1 of Example 4 of the present invention; A is 13 C. Carbon spectrum; B is 1 H is hydrogen spectrum; C is HSQC spectrum; D is 1 H- 1 H COSY spectrum; E is HMBC spectrum;

[0035] Figure 10 Graph showing the inhibitory effect of Qihong polysaccharide KBPW1 on CT26 WT colorectal cancer cells in Example 5 of the present invention; A shows the inhibitory effect of Qihong polysaccharide KBTP-1 on CT26-WT cell proliferation; B shows the migration of cells scratched after Qihong polysaccharide was treated with different concentrations of KBTP-1 for 0, 24, and 48 hours;

[0036] Figure 11 This is a graph showing the tumor inhibition effect of Qihong polysaccharide KBPW1 in Example 6 of the present invention on CT26 WT colorectal cancer-bearing mice. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0038] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0039] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0040] Example 1: Response surface optimization of Qihong polysaccharide extraction process

[0041] (1) Remove impurities, take an appropriate amount of tea leaves, grind them with a grinder, sieve them through an 80-mesh sieve, and store them in a dark, dry place for later use; accurately weigh the tea powder, add 95% ethanol at a material-liquid ratio of 1:20 g / mL, stir at 25°C and 120 rpm / min for 24 h, filter, collect the precipitate, and dry it in a 55°C oven to obtain decolorized and defatted dry tea powder;

[0042] (2) Single factor experiment, respectively set the solid-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50 g / mL), time (1, 1.5, 2, 2.5, 3 h), temperature (60, 70, 80, 90, 100 ° C) and other factors for extracting Qihong polysaccharide. Taking the yield of Qihong polysaccharide as the index, the influence of each factor variable on the extraction rate of Qihong polysaccharide was investigated to determine the optimal extraction process parameters.

[0043] (3) Response surface optimization design: The factors and levels of the response surface design were determined by the optimal point of the single factor experiment. The Box-Benhnken L 17 (33 ) designed an experimental plan, using the solid-liquid ratio, temperature, and time for extracting Qihong polysaccharides as dependent variables and polysaccharide yield as the variable for response surface analysis. Each independent variable level was coded as -1, 0, or 1, respectively. The experimental factors and levels are shown in Table 1.

[0044] Table 1 Coding levels of independent variables in response surface experiments

[0045]

[0046]

[0047] (4) Single factor experimental results:

[0048] like Figure 1 A shows that with the increase of the solid-liquid ratio, the extraction rate of Qihong polysaccharides shows an upward trend, and the extraction rate reaches the highest value at a solid-liquid ratio of 1:30g / mL. However, when the solid-liquid ratio continues to increase, the polysaccharide yield begins to decline. It is speculated that when the solid-liquid ratio is low, the polysaccharide is not fully swollen, and the extract is difficult to separate, affecting the precipitation of polysaccharides. When the solid-liquid ratio is high, the solution becomes dilute, and the adsorption between molecules is enhanced, resulting in a decrease in the polysaccharide extraction rate and a larger solvent, which increases the subsequent processing cost. Therefore, the optimal solid-liquid ratio is 1:30g / mL.

[0049] Figure 1 Figure B shows that with increasing extraction temperature, the yield of Qihong polysaccharides initially increases and then decreases, reaching its maximum yield at 90°C. The diffusion coefficient of polysaccharides increases with increasing extraction temperature, leading to an increase in their content in the aqueous solution. However, excessively high temperatures can cause polysaccharide degradation, resulting in a decrease in yield and increased energy consumption. Therefore, the optimal extraction temperature is 90°C.

[0050] Figure 1 C shows that the polysaccharide extraction rate slowly increases with increasing extraction time. The maximum polysaccharide yield, 5.35 ± 0.35%, is reached at 2.5 hours. However, after 2.5 hours, the yield begins to decline. This is because extending the extraction time facilitates and accelerates the dissolution of polysaccharides into the solvent. However, excessive extraction time can also lead to polysaccharide degradation, reducing yield. Therefore, the optimal extraction time is 2.5 hours.

[0051] (5) Response surface test results: Based on the single factor test, liquid-to-solid ratio, extraction temperature, and extraction time were used as experimental factors to conduct L 17 (3 3) response surface experiment was conducted to optimize the extraction process of Qihong polysaccharide. The experimental design and results are shown in Table 2. The data in Table 2 were subjected to multiple regression fitting using Design-Expert 13.0 software, and the quadratic multiple regression equation of Qihong polysaccharide yield (Y) versus solid-liquid ratio (A), temperature (B), and time (C) was obtained:

[0052] Y=7.48+0.06400A+0.3563B+0.2013C-0.1750AB-0.0450AC+0.1475BC-0.6878A 2 -0.91

[0053] 53B 2 -1.01C 2 .

[0054] Table 2 Factor levels and results of response surface experiment

[0055]

[0056]

[0057] Table 3 Analysis of variance

[0058]

[0059] Note: *p<0.05 indicates significant difference; **p<0.01 indicates extremely significant difference

[0060] The above regression mathematical model was subjected to variance analysis to test the validity of the equation and the partial regression coefficients of each factor. As shown in Table 3, the model selected in this experiment is extremely significant (P<0.01), and the lack of fit term of the variance is not significant (P=0.5787>0.05), indicating that non-experimental factors have little effect on the test results. In addition, the coefficient of determination of the selected model R 2 The value is 0.9760, which shows that the model fits the actual experiment well. The corrected determination coefficient R 2 The Adj value is 0.9451, which is consistent with R 2 The values are close, indicating that the model has sufficient accuracy and versatility. According to the F value, it can be seen that the influence of various factors on the polysaccharide yield is: material-liquid ratio> temperature> time. It can also be seen from the table that A, B, A 2 、B 2 、C 2 The effect of C on polysaccharide yield was extremely significant (P<0.01), and C had a significant effect on polysaccharide yield (P<0.05); the effects of other factors were not significant.

[0061] The response surface and contour plots of the interactions between the factors in the regression model are as follows: Figure 2As shown in the figure, the optimal extraction process for crude Qihong polysaccharides obtained by the regression model is: solid-liquid ratio of 1:34.418 g / mL, temperature of 91.6057°C, and extraction time of 2.55097 h. Under these conditions, the polysaccharide extraction rate reached 7.65811%. Considering feasibility from a practical perspective, based on the model results and actual conditions, the optimal extraction parameters were set as: solid-liquid ratio of 1:30 g / mL, temperature of 90°C, and extraction time of 2.5 h. The average yield of crude polysaccharides was 7.48 ± 0.22%, which is basically consistent with the predicted results. The results show that this method can be used to obtain the optimal process parameters for polysaccharide extraction.

[0062] Example 2: Preparation and purification of Qihong polysaccharide

[0063] (1) The decolorized and defatted tea powder obtained in Example 1 was mixed uniformly with distilled water at a material-liquid ratio of 1:30 g / mL, stirred and extracted at 80°C and 120 rpm / min for 2.5 h, filtered, and the filtrate was collected. This was repeated twice, and the filtrates from the three extractions were combined to obtain a tea extract.

[0064] (2) The tea extract was concentrated to 1 / 4 of its original volume in a rotary evaporator (35 rpm / min, 55°C, 0.09-0.10 MPa) under reduced pressure. The extract was centrifuged at 4°C and 10,000 rpm / min for 10 min, the precipitate was discarded, and the supernatant was collected. Four volumes of anhydrous ethanol (to a final ethanol concentration of 80%) were added to the collected concentrate. After precipitation (at room temperature) for 12 h, the precipitate was collected by gauze filtration and dried in a 55°C oven to obtain a tea alcohol precipitate.

[0065] (3) Take the tea alcohol precipitate, add an appropriate amount of deionized water (100 mL) until it is completely dissolved, add 1 / 4 volume (25 mL) of Sevag reagent (chloroform / n-butanol = 4 / 1), and stir magnetically at room temperature for 30 minutes. Repeat 4 times until there is no white protein layer, and collect the supernatant polysaccharide solution; take the polysaccharide solution after deproteinization and place it in a petri dish, seal it with plastic wrap, and freeze it in a -20°C refrigerator for 12 hours, and then freeze-dry it in a vacuum freeze dryer (-50°C, 10 Pa) for 48 hours to obtain a tea polysaccharide sample;

[0066] (4) Using tea polysaccharide sample, prepare 20 mg / mL tea polysaccharide solution, elute through DEAE-Sepharose Fast Flow ion exchange column, use deionized water and 0.1, 0.2, 0.3, 0.4, 0.5 mol / L NaCl solution in sequence for gradient elution at a flow rate of 5 mL / min, detect the polysaccharide content in the eluate by phenol-sulfuric acid colorimetry, collect the sugar-containing eluate, obtain Qihong polysaccharide extract, and draw the elution curve, as shown in Figure 4. Figure 3 A.

[0067] (5) The deionized water eluate of the main component was collected and further purified using a Sephadex G-100 gel column, eluted with deionized water at a flow rate of 0.5 mL / min, and the polysaccharide content in the eluate was detected by phenol-sulfuric acid colorimetry. The elution curve was drawn, as shown in FIG. Figure 3 B. The polysaccharide-containing eluate was collected, concentrated under reduced pressure, dialyzed for desalination, and vacuum freeze-dried to obtain the Qihong polysaccharide fraction KBPW1.

[0068] Example 3: Physicochemical properties and structural characterization of Qihong polysaccharide KBPW1

[0069] (1) Determination of carbohydrate content in Qihong polysaccharide KBPW1

[0070] The carbohydrate content of KBPW1 obtained in Example 1 was determined by the phenol-sulfuric acid method. Detection by spectrophotometer at 490 nm showed that the carbohydrate content of KBPW1 was 83.60±0.79%.

[0071] (2) UV spectroscopy analysis of Qihong polysaccharide KBPW1

[0072] Take Qihong polysaccharide KBPW1 and prepare it into 0.1 mg / mL solution. Scan it in the wavelength range of 190-400 nm using a full wavelength scanning microplate reader. Figure 4 A, KBPW1 has no absorption peaks at 260 nm and 280 nm, indicating that KBPW1 contains almost no pigment, protein, and nucleic acid.

[0073] (3) Determination of the uniformity and weight-average molecular weight of Qihong polysaccharide KBPW1

[0074] High performance gel permeation chromatography (HPGPC) was used to determine the retention time of dextran standards with different molecular weights (5KD, 25KD, 80KD, 150KD, 420KD, 670KD), and a molecular weight standard curve was drawn. Then, the relative molecular weight was calculated based on the standard curve and the peak time of the Qihong polysaccharide KBPW1 sample.

[0075] HPGPC detection conditions: chromatographic column: BRT105-103-101 series gel column (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.8 mL / min; column temperature: 40°C; injection volume: 25 μl; detector: differential detector RID-20A.

[0076] The results are as follows Figure 4 As shown in B, Qihong polysaccharide KBPW1 is a homogeneous polysaccharide with a weight average molecular weight of 3.8×10 3 Da.

[0077] (4) Analysis of the morphology of Qihong polysaccharide KBPW1

[0078] The surface morphology of Qihong polysaccharide KBPW1 was analyzed by scanning electron microscopy. 2 mg of KBPW1 was weighed and adhered to a metal iron block with conductive glue. A layer of gold was sprayed on the surface of the polysaccharide sample using a spraying device, and then the surface morphology was observed using a scanning electron microscope. The results are as follows. Figure 6 As shown, the surface of KBPW1 is uniform and smooth, presenting irregular lamellar shapes.

[0079] (5) Characteristic group analysis of Qihong polysaccharide KBPW1

[0080] The characteristic groups of Qihong polysaccharide KBPW1 were analyzed by Fourier transform infrared spectrometer. 5 mg of Qihong polysaccharide KBPW1 and 100 mg of potassium bromide were weighed and mixed evenly. After grinding, drying, and pressing, the tablets were placed on Nicolet 67 Fourier transform infrared spectrometer for scanning analysis with a scanning range of 4000 cm -1 -500cm -1 The results are as follows Figure 5 As shown, Qihong polysaccharide KBPW1 has a typical characteristic peak of polysaccharide, 3405.19 cm -1 A strong absorption peak is generated at 2923.07cm, which is the stretching vibration of -OH. -1 The absorption peak at 1617.33 cm is caused by the stretching vibration of CH in the CH2 group. -1 The absorption peak at 1401.24 cm is the stretching vibration of C=O. -1 The absorption peak at 1100.29 cm is related to the CH bending vibration; -1 The strong absorption peak at 963.75 cm is attributed to the stretching vibration of the pyran ring of the glycosyl residue. -1 and 640.73cm -1 The absorption peak formed at is related to the presence of β-glycosidic bonds and α-glycosidic bonds in the polysaccharide.

[0081] (6) Analysis of monosaccharide composition of Qihong polysaccharide KBPW1

[0082] The monosaccharide composition of the Qihong polysaccharide KBPW1 was determined using a Thermo ICS5000 ion chromatograph. 5 mg of KBPW1 was precisely weighed and placed in an ampoule. 2 mL of 3 mol / L trifluoroacetic acid (TFA) was added and hydrolyzed at 120°C for 3 h. The acid hydrolysis solution was accurately pipetted and transferred to a tube. After nitrogen evaporation, 5 mL of ultrapure water was added and vortexed to mix thoroughly. The tube was then centrifuged at 12,000 rpm for 5 min. The supernatant was transferred to a chromatographic vial for analysis. Separately, 5 mg of each of the following monosaccharide standards were accurately weighed: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid. Monosaccharide standard solutions with varying concentration gradients were prepared and the masses of the different monosaccharides were determined using absolute quantification methods. The molar ratios were calculated based on the molar masses of the monosaccharides.

[0083] IC detection conditions: Column: Dionex Carbopac TM PA20 (3*150mm); mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaOAc; flow rate: 0.3mL / min; injection volume: 25μL; column temperature: 30℃; elution gradient: 0min phase A / phase B / phase C (98.8:1.2:0, V / V), 18min phase A / phase B / phase C (98.8:1.2:0, V / V), 20min phase A / phase B / phase C (50:50:0, V / V), 30min phase A / phase B / phase C (50:50:0, V / V), 30.1min phase A / phase B / phase C (0:0:100, V / V), 46min phase A / phase B / phase C (0:0:100, V / V), 46.1min Phase A / B / C (0:100:0, V / V), 50 min Phase A / B / C (0:100:0, V / V), 50.1 min Phase A / B / C (98.8:1.2:0, V / V), 80 min Phase A / B / C (98.8:1.2:0, V / V). Detector: Electrochemical detector.

[0084] The results are as follows Figure 7 As shown, the monosaccharide composition of KBPW1 is arabinose Ara, galactose Gal, and glucose Glc, with a molar ratio of 2.62:1.02:6.36.

[0085] Example 4: Structural Identification of Qihong Polysaccharide KBPW1

[0086] (1) Analysis of glycosidic bond composition of Qihong polysaccharide KBPW1

[0087] The glycosidic bond type of KBPW1 was analyzed by gas chromatography-mass spectrometry (GC-MS). 20 mg of KBPW1 was placed in a glass reaction bottle, and 1 mL of anhydrous DMSO was added. According to the instructions of the methylation kit (BRT-JJH), methylation reagent A solution was added for sealing. After ultrasonic dissolution, methylation reagent B solution was added. After stirring in a 30°C water bath for 60 minutes, 2 mL of ultrapure water was added to terminate the methylation reaction. Finally, the reaction solution was concentrated, dialyzed, and freeze-dried to obtain a completely methylated polysaccharide sample.

[0088] The methylated polysaccharide sample was placed in an ampoule, 1 mL of TFA (2 mol / L) was added, and the sample was hydrolyzed at 110°C for 90 min. The TFA was completely removed by rotary evaporation to obtain a completely hydrolyzed product. 2 mL of double-distilled water and 60 mg of sodium borohydride (NaBH4) were added to the hydrolyzate for reduction reaction. After 8 h, glacial acetic acid was added to neutralize the sample. After rotary evaporation to remove excess NaBH4, 1 mL of acetic anhydride was added and the sample was reacted at 100°C for 1 h. After cooling, 3 mL of toluene was added, and the excess acetic anhydride was evaporated under reduced pressure. The acetylated product was extracted with dichloromethane (CH2Cl2). The extract was dried over anhydrous sodium sulfate and analyzed by GC-MS.

[0089] GC-MS detection conditions: Agilent 7890A-5977B system, chromatographic column: RXI-5SIL MS (30m*0.25mm*0.25um); programmed temperature conditions: starting temperature 120℃, heating to 250℃ / min at 3℃ / min; holding for 5min; inlet temperature: 250℃, detector temperature: 250℃, carrier gas: helium, flow rate: 1mL / min; ion flame detector, mass scan range (m / z): 50-350.

[0090] The results are as follows Figure 8 As shown in Table 4, the methylated product of KBPW1 showed eight methylated derivative ion peaks in the gas chromatogram. According to the retention time of each peak, the glycosidic bond type and molar ratio of Qihong polysaccharide KBPW1 were obtained by online retrieval of each mass spectrum.

[0091] (2) Analysis of glycosidic bond connection mode of Qihong polysaccharide KBPW1

[0092] The glycosidic bond connection mode of KBPW1 was analyzed by nuclear magnetic resonance: 50 mg of KBPW1 was dissolved in 1 mL of heavy water, the solution was transferred into a nuclear magnetic resonance tube, and the sample was scanned using an Agilent VNMRS600 superconducting nuclear magnetic resonance spectrometer. 1 H-NMR, 13 C-NMR spectra and two-dimensional 1 H- 1H COSY, HMBC, HSQC spectra ( Figure 9 ).

[0093] As shown in Figure 9, the sugar residues of KBPW1 13 C. 1 The chemical shift assignments for H are shown in Table 5, which are generally consistent with the monosaccharide composition and methylation structure. Based on the above structural characterization of KBPW1, the chemical structure of KBPW1 according to the present invention can be derived as follows:

[0094]

[0095] Table 4 Glycosidic bond types of KBPW1

[0096] Serial number Retention time (min) Glycosyl derivatives molar ratio Glycosidic bond connection 1 10.412 <![CDATA[2,3,5-Me3-Araf]]> 16.2 Araf-(1→ 2 15.098 <![CDATA[2,3-Me2-Araf]]> 8.5 →5)-Araf-(1→ 3 16.862 <![CDATA[2,3,4,6-Me4-Glcp]]> 4.8 Glcp-(1→ 4 18.412 <![CDATA[2-Me1-Araf]]> 7.2 →3,5)-Araf-(1→ 5 20.768 <![CDATA[2,3,6-Me3-Glcp]]> 45.5 →4)-Glcp-(1→ 6 21.322 <![CDATA[2,4,6-Me3-Galp]]> 11.4 →3)-Galp-(1→ 7 22.06 <![CDATA[2,3,4-Me3-Glcp]]> 2.6 →6-Glcp-(1→ 8 26.55 <![CDATA[2,3-Me2-Glcp]]> 3.8 →4,6)-Glcp-(1→

[0097] Table 5 Glycosidic bonds 1 H and 13 Chemical shift of C

[0098]

[0099]

[0100] Example 5: In vitro inhibition of migration of CT26 WT colorectal cancer cells by Qihong polysaccharide KBPW1

[0101] (1) Cell proliferation assay

[0102] The effect of Qihong polysaccharide KBPW1 prepared in Example 1 on the activity of mouse colorectal cancer cells CT26 WT was detected by CCK8. CT26.WT cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 5×10 4 / mL, seeded in a 96-well plate, 100 μL of cell suspension per well, placed in a 37°C, 5% CO2 incubator for 24 hours of adherent culture, then added with KBPW1 at final concentrations of 50, 100, 200, 400, 600, and 800 μg / mL, respectively. A blank culture medium control group was also set up. After further incubation for 24 hours, CCK-8 (10 μL) was added to each well and incubated for 2 hours. Finally, the OD value of each well was measured at a wavelength of 450 nm using a microplate reader to calculate the effect of Qihong polysaccharide KBPW1 on tumor cell viability. Cell viability (%) = (OD drug group - OD blank group) / (OD control group - OD blank group) × 100%, with the cell viability of the control group set to 100%.

[0103] The results are as follows Figure 10As shown in A, after CT26.WT cells were incubated with Qihong polysaccharide KBPW1, the inhibitory ability of KBTP-1 on CT26-WT cells increased with the increase of KBPW1 concentration, and the inhibitory activity was dose-dependent. When the KBPW1 concentration was 800 μg / mL, the cell survival rate was less than 50%, proving that Qihong polysaccharide KBPW1 can inhibit the proliferation of colorectal cancer cells CT26 WT to a certain extent.

[0104] (2) Cell scratch test: CT26-WT cells were plated at 1×10 6 Cells were seeded at a density of 100 μg / mL in a 6-well plate. After 24 hours of adherence, a uniform vertical scratch was made in the center of each well using a 200 μL pipette tip. The cells were then cultured in serum-free medium containing 100, 200, or 400 μg / mL of KBPW1. Changes in the scratch pattern were observed under an inverted fluorescence microscope at 0, 24, and 48 hours of culture to record changes in the migration ability of CT26-WT cells.

[0105] The experimental results are as follows Figure 10 As shown in Figure B, it can be seen that after incubation with KBPW1, the migration ability of CT26-WT cells was greatly reduced. Moreover, the cell migration ability decreased with the increase of KBPW1 concentration, indicating that KBPW1 can significantly inhibit the migration of colon cancer cells CT26-WT, and the inhibitory effect is dose-dependent.

[0106] Example 6: Study on the anti-tumor activity of Qihong polysaccharide KBPW1 in vivo

[0107] A tumor-bearing mouse model was established by xenografting colorectal cancer CT26 WT cells. Different doses of Qihong polysaccharide KBPW1 were orally administered to analyze the antitumor activity of KBPW1.

[0108] (1) Construction of colorectal cancer tumor-bearing mouse model and drug intervention

[0109] Five-week-old male BALB / C mice were acclimated in an SPF animal room at 23±2°C, 50%-60% relative humidity, 12 h light and 12 h dark conditions for one week. Seventy-two mice were randomly selected. Except for the normal group (Normal, 12 mice), the remaining mice were inoculated with CT26 WT colorectal cancer cells. A 1 mL microsyringe was used to extract a suspension of CT26 WT cells in the logarithmic growth phase (2×10 6 The cells / mL) were inoculated into the right axilla of 60 sterilized BALB / C mice, with each mouse receiving 0.2 mL (2×10 6The injection site was compressed to prevent cell leakage. Mice bearing CT26.WT colorectal cancer cells were randomly divided into five groups (n=12) after inoculation and administrated with sterile saline. The following groups were randomly assigned: model group (gavage with sterile saline); positive group (gavage with 5-fluorouracil, 20 mg / kg·dBW); low-dose KBPW1 group (KBPW1-L, 50 mg / kg·d BW); medium-dose KBPW1 group (KBPW1-M, 100 mg / kg·d BW); and high-dose KBPW1 group (KBPW1-H, 200 mg / kg·d BW). Sterile saline was used as the solvent for the Positive and KBPW1 groups. Each group received the drug by oral gavage once daily at a volume of 0.1 mL / 10 g for 15 consecutive days. Mice were then sacrificed by CO2 asphyxiation and the relevant indicators were measured.

[0110] (2) Anti-tumor activity detection indicators in mice

[0111] General observation: Observe the mice's mental state, activity, hair glossiness and other routine basic indicators every day.

[0112] Body weight and diet and water intake testing: During the experiment, from the 0th day after gavage to the 15th day after gavage, the body weight, diet and water intake of each group of mice were monitored and recorded every 3 days, and the body weight and diet change curves were drawn.

[0113] Tumor volume determination: During oral administration, the long and short diameters of the mouse tumor were measured with a vernier caliper every 3 days, and the tumor growth of the mouse was monitored and recorded regularly; according to the formula V = 0.5ab 2 (a-long diameter of the tumor, b-short diameter of the tumor) Calculate the tumor volume, draw the tumor volume change curve, and calculate the relative tumor volume and relative tumor proliferation rate.

[0114] Tumor inhibition rate determination: After the oral administration, the axillary tumors of each group of mice were removed by dissection, weighed, measured, and photographed to calculate the tumor inhibition rate. Tumor inhibition rate % = (1-average tumor weight of the drug administration group / average tumor weight of the model control group) × 100%

[0115] Determination of organ index: After the experiment, the thymus of the mouse was removed, rinsed with physiological saline, dried with filter paper and weighed in sequence. Thymus index = (thymus weight / animal body weight) × 100%.

[0116] (3) Experimental results

[0117] The in vivo experiment of Qihong polysaccharide KBPW1 against CT26 WT colorectal cancer-bearing mice showed that the volume and weight of the tumor mass in the right axilla of the model control group continued to expand over time, while the KBPW1-treated group could significantly inhibit the growth of tumors in the tumor-bearing mice, and its tumor inhibition effect was concentration-dependent. Although the 5-FU-positive group showed a high tumor inhibition activity, it had obvious side effects on the mice, resulting in weight loss, decreased immune organ index, significantly reduced vitality, and poor physiological condition (Table 6 and Figure 11 ). Statistical analysis of the tumor inhibition rates of different groups was further performed. The results showed that the high-dose group of Qihong polysaccharide KBPW1 achieved a tumor inhibition rate of 71.26±4.34% in CT26 WT colorectal cancer-bearing mice, and significantly improved the thymus index of the immune organ of tumor-bearing mice. The above results indicate that Qihong polysaccharide KBPW1 has a good in vivo anti-CT26 WT colorectal cancer effect, and its toxic side effects are far less than those of the traditional anti-tumor drug 5-FU, with little effect on the physiological state of mice, and no adverse reactions with long-term use.

[0118] Table 6 Inhibition of tumor growth in CT26 WT colorectal cancer-bearing mice by Qihong polysaccharide KBPW1

[0119]

[0120] Note: The data in the table are ** P<0.01, * P < 0.05, compared with the model group; ## P<0.01, # P < 0.05, compared with the normal group.

[0121] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A Qihong polysaccharide KBPW1 with anti-tumor activity, characterized in that: The repeatable primary structural unit of the Qihong polysaccharide KBPW1 contains →4)-β-D-Glc p -(1→、α-L-Ara f -(1→、→3)-β-D-Gal p -(1→、→5)-α-L-Ara f -(1→、→3,5)-β-L-Ara f -(1→,β-D-Glc p -(1→、→4,6)-β-D-Glc p -(1→、→6)-β-D-Glc p -(1→eight glycosidic bonds; the connection mode of the glycosidic bonds is shown in Formula 1: Formula 1; The Qihong polysaccharide KBPW1 consists of arabinose, galactose and glucose, and the molar ratio of arabinose, galactose and glucose is 2.62:1.02:6.

36.

2. The Qihong polysaccharide KBPW1 with anti-tumor activity according to claim 1, characterized in that: The relative molecular mass of the Qihong polysaccharide KBPW1 is 3.8×10 3 Da.

3. Use of the Qihong polysaccharide KBPW1 with anti-tumor activity according to any one of claims 1-2 in the preparation of anti-colorectal cancer drugs.

4. An anti-colorectal cancer pharmaceutical composition, characterized in that: The ingredients include the Qihong polysaccharide KBPW1 with anti-tumor activity as described in any one of claims 1-2.

5. The anti-colorectal cancer pharmaceutical composition according to claim 4, characterized in that: Its components also include one or more pharmaceutically acceptable carriers.

6. The anti-colorectal cancer pharmaceutical composition according to claim 5, characterized in that The pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorption carrier, surfactant or lubricant.

7. The anti-colorectal cancer pharmaceutical composition according to claim 6, characterized in that: The pharmaceutically acceptable carrier is a diluent.

8. The anti-colorectal cancer pharmaceutical composition according to claim 4, characterized in that: The oral dosage forms of the pharmaceutical composition include tablets, capsules, oral liquids, granules, pills or powders.

9. The anti-colorectal cancer pharmaceutical composition according to claim 8, characterized in that: The oral dosage form of the pharmaceutical composition is a tablet.

10. The anti-colorectal cancer pharmaceutical composition according to claim 8, characterized in that: The oral dosage form of the pharmaceutical composition is granules.

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